Condensate colorimetric nitrogen oxide analyzer

ABSTRACT

Disclosed is a device for determination of the content of higher oxides of nitrogen in exhaled breath condensate which comprises a conduit having an exhalate condensing portion with an inlet and an outlet (the inlet can be configured to fit with a mechanical respirator or, for direct use by the patient, an inlet assembly providing one-way ingress of ambient atmosphere to the device can be associated with the inlet of the conduit exhalate condensing portion); a coolant jacket coaxially surrounding said exhalate condensing portion; a gas release port; and, in enclosed fluid communication with said conduit exhalate condensing portion outlet, a sample assay assembly comprising (i) a translucent analysis chamber attached to the outlet to receive condensate fluid and having a reagent entry port, (ii) a reagent chamber in enclosed fluid communication with the reagent entry port and (iii) a pliable element connecting the outlet and the analysis chamber and forming a portion of the reagent chamber such that flexion of the pliable element closes communication between the outlet and the analysis chamber and contracts the reagent chamber so as to deliver to the analysis chamber controlled amounts of condensate and reagent. The device is disposable and inexpensive, and is used to collect human exhalate for colorimetric assay of liquid and gas phase nitrogen oxides to assist in evaluation of airway inflammation.

This is a continuation of application Ser. No. 08/623,643, filed Mar.28, 1996, now U.S. Pat. No. 6,033,368.

FIELD OF THE INVENTION

The invention is directed to a condensate colorimetric nitrogen analyzeroxide that determines the content of nitrogen oxides in exhaled breathcondensate.

BACKGROUND OF THE INVENTION

Conventional assessment of severity of airway diseases, includingasthma, consists of measures of pulmonary mechanics which are effortdependent and thus not suitable for many pediatric patients,mechanically ventilated patients, or patients with neuromusculardisease. See Crapo R., Pulmonary-function testing. N. Engl. J. Med.,331; 1:25-30; 1994; and American Thoracic Society/European RespiratorySociety: Respiratory mechanics in infants—physiologic evaluation inhealth and disease, Am. Rev. Respir. Dis., 1993; 147:474-96. Currentlythe Peak Expiratory flow meter is the only method commonly available forhome use by asthmatics to monitor the state of their airway disease.This test suffers from being an insensitive indicator of airwayinflammation, and is effort dependent. Other physiologic parametersrequire expensive machines and experienced operators to obtainreproducible results. Klein R., Fritz G. Yeung A., McQuade E., MansellA., Spirometric Patterns in Childhood Asthma: Peak Flow Compared withOther Indices. Pediatr. Pulmonol., 1995, 20:372-379.

Endogenous production of nitrogen oxide in the human airway has beenshown to be increased in patients with asthma and other inflammatorylung diseases. Gaston B., Drazen J., Chee, C., et al. Expired nitricoxide concentrations are elevated in patients with reactive airwaysdisease. Endothelium. 1993; 1:S87; Gustafsson, I. E., Leone A. M.,Persson M. G., Wiklund N. P., Moncada S., Endogenous nitric oxide ispresent in the exhaled air of rabbits, guinea pigs and humans. 1991;181; 2:852-857. However, measurement of gas phase nitric oxide (nitrogenmonoxide) per se in the parts per billion range found in the airwayrequires the use of mass spectrometry or chemiluminescent technology,which are cumbersome, expensive and not reasonably suited for home use.

Moreover, measurement of aqueous phase higher oxides of nitrogencompounds such as S-nitrosothiols, nitrite and nitrate, which are notnitrogen monoxide but which are likewise elevated in concentrations insubjects with inflammatory lung disease, has required that patients behospitalized for invasive airway sampling procedures, including airwayintubation and bronchoalveolar lavage. Gaston, B., Reilly J., Drazen J.,et al., Endogenous nitrogen oxides and bronchodilator S-nitrosothiols inhuman airways. Proc. Natl. Acad. Sci., 1993; 90:10957-10961.

SUMMARY OF THE INVENTION

The invention provides a device for determination of the content ofhigher oxides of nitrogen in exhaled breath condensate which comprises aconduit having an exhalate condensing portion with an inlet and anoutlet (the inlet can be configured to fit with a mechanical respiratoror, for direct use by the patient, an inlet assembly providing one-wayingress of ambient atmosphere to the device can be associated with theinlet of the conduit exhalate condensing portion); a coolant jacketcoaxially surrounding said exhalate condensing portion; a gas releaseport; and, in enclosed fluid communication with said conduit exhalatecondensing portion outlet, a sample assay assembly comprising (i) anoptical analysis chamber, for example, clear or translucent, attached tothe outlet to receive condensate fluid and having a reagent entry port(ii) a reagent chamber in enclosed fluid communication with the reagententry port and (iii) a pliable element connecting the outlet and theanalysis chamber and forming a portion of the reagent chamber such thatflexion of the pliable element closes communication between the outletand the analysis chamber and contracts the reagent chamber so as todeliver to the analysis chamber controlled amounts of condensate andreagent. The device is disposable and inexpensive, and is used tocollect human exhalate for colorimetric assay of liquid and gas phasenitrogen oxides to assist in evaluation of airway inflammation.

In a preferred embodiment the device further comprises an inlet assemblyproviding one-way ingress of ambient atmosphere to the exhalatecondensing conduit inlet. This is ideal for the patient who is breathingdirectly into the device, rather than the situation where the device isconnected to a mechanical respirator.

In a preferred embodiment the device further comprises, between theinlet assembly and the exhalate condensing conduit portion, a filtercapable of removing particulate matter from exhaled air which passestherethrough.

In another preferred embodiment, the device further comprises a coolantmaterial in the coolant jacket.

In another preferred embodiment, the device further comprises a reagentcomposition capable of detecting a nitrite or nitrate in an exhalationcondensate sample.

In another preferred embodiment, the device further comprises areagent-permeable membrane in the reagent entry port.

In another preferred embodiment, the device further comprises arupturable membrane in the reagent entry port.

In another preferred embodiment, the inlet assembly further includestherein a filter capable of removing ambient nitrogen oxides fromexhalate passing therethrough.

In another preferred embodiment the exhalate condensing conduit portioncomprises a single lumen conduit having a lumen diameter sufficient tocause substantially no resistance to the flow of exhalate therethrough.

In another preferred embodiment the exhalate condensing conduit portioncomprises a plurality of conduit tubules having a collective lumendiameter sufficient to cause substantially no resistance to the flow ofexhalate therethrough.

The purpose of this invention is to condense exhaled lung gas and vaporfor aqueous phase nitrogen oxide analyses. This will assist in theevaluation of the human airways' production of nitrogen oxides in bothgas and several liquid phase states, including nitrite and nitrate, withpotential for multiple other studies to help delineate the lung's redoxenvironment and the airways' degree of inflammation. The investigationswhich have resulted in the present invention indicate that easilymeasured liquid phase exhaled nitrogen oxides, nitrite and nitrate, arelikewise elevated in asthmatic subjects during periods of inflammation(see abstract enclosed, unpublished data). Utility in the clinicalsetting for inexpensive and domiciliary evaluation of airwayinflammation in various disease states, including asthma, is the primarypurpose of the device of the present invention and its utility has beenproven, inter alia, as demonstrated herein.

The invention is comprised of cold tolerant materials and consists oftwo tubing units, one inside the other. Surrounding the inner tube orset of tubes, and contained by the outer tube, is a chemical substancewhich has a high specific heat, and therefore, once frozen, can maintainfreezing temperatures for an extended period of time. The disposableunit can be frozen in a standard size home freezer and then connectedtogether with collection and analysis instruments as a compact integralunit.

Attached to the proximal portion of the unit is a port through which thesubject breathes. This consists of two one-way valves which directatmospheric air or selected gases to the patient's lungs duringinspiration, and channel exhaled gas down a condensing tube. Gas movesin only one direction through the condensing apparatus. Inserted betweenthe breathing port's mouthpiece and the condensing chamber is amicroporous filter which traps all small particles (such as saliva orsputum), is impermeable to liquids, but allows gas and vaporized fluidsof less than 0.3 microns in diameter to pass. This acts as a saliva trapand may also act as a filter for the larger fluid particles which may beaerosolized in the larger airways.

The distal end of the condensing chamber tube(s) is attached to acollecting apparatus which utilizes gravity to trap condensed fluid. Atthe bottom of this trap is a clear plastic analyzing chamber in whichthe sample is sequentially warmed, sealed, reacted with colorimetricreagents and analyzed spectrophotometrically by the non-disposablecolorimeter.

The patient breathes comfortably in and out through the mouthpiece. Lungfluid vapor collects on the inner surface of the inner tube(s) of thecondensing apparatus starting immediately. Gravity carries the largerdroplets down the tube, these droplets recruiting other small dropletson their trip to the collecting vial distally. Alternatively, after afixed period of tidal breathing, the condensed fluid can be expresseddown the inner tube with a device similar to a syringe plunger. Aqueousphase nitrite and nitrate can be measured by standard colorimetricassays, and can be reasonably quantified by simple tests performed bypatients in their homes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a side elevation view, partially in section, of the device ofthe invention.

FIG. 2A is a side sectional elevation view of a preferred embodiment ofthe sample assay assembly of the device with the non-disposableanalyzer, and includes a top view of the connecting pincer valve.

FIG. 2B is a top view of the pincer which flexes the pliable element ofthe sample assay assembly.

FIG. 3 is a histogram showing the results of the nitrite assay performedas described in Example 2.

FIG. 4 is a histogram showing the results of the nitrate assay performedas described in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

The device of the invention has several advantages, including:

1. This is the only device which measures airway inflammation with asimple home assay, without invasive procedures or expensive andcumbersome testing equipment.

2. Ease of transportability and use of the apparatus.

3. Clinical application for individual patients in their own home or inthe clinic or hospital setting to test for or monitor status of asthmaor other causes of airway inflammation. May be used to predict diseaseexacerbations, allowing time for successful early intervention.

4. Clinical applicability in the setting of mechanically ventilatedpatient to easily monitor airway inflammation.

5. Clinical applicability for measuring airway inflammation in manychildren too young to cooperate with standard pulmonary functiontesting, but who can cooperate with tidal breathing through a tube.Similar potential in patients with neuromuscular disease in whomstandard tests are uninterpretable.

The device can be made with easily available materials. The coolingsubstance in the condensing chamber can be water, or a viscous substancewith a high specific heat. The device can also be made, as describedabove, without refrigerant, utilizing a gas/membrane or gas/aqueousreaction compartment.

Referring now to FIG. 1, mouthpiece 1 is formed as an integral elementwith low resistance one-way valve 2 allowing inspiration of air into thedevice of the invention resulting from inhalation through it by theuser. Low resistance one-way valve 2 optionally contains potassiumpermanganate and a charcoal filter to remove ambient nitrogen oxides.Mouthpiece 1 and low resistance one-way valve 2 are further integralwith low resistance one-way valve 3 to direct all expired gas from theuser into the remaining elements of the device. Integral with anddownstream of low resistance one-way valve 3 is low resistance filter 4,containing elements which removes all particulates greater than 0.03 μMin diameter.

Connected downstream to low resistance filter 4 is condensing chamber 5which consists of either a single, long inert plastic tube of sufficientdiameter to be of low resistance to air expired into the device, or abundle of shorter, parallel, inert plastic tubes of sufficient number toprovide minimal resistance to airflow. Condensing chamber 5 is incontinuity with, i.e., receives air arising from filter 4. Coolantchamber or jacket 6 contains an aqueous solution of sufficientosmolality to cause freezing point depression to just above the minimumtemperature of the average household freezer, e.g., in a range of ±15°F., preferably below 0° F.

Condensing chamber 5 is completely sealed from exposure to coolantchamber 6. Essentially coolant chamber 6 coaxially surrounds condensingchamber 5 so as to control the temperature within condensing chamber 5.Condensing chamber 5 is continuous with a conduit that divides tocondensate delivery conduit 7 and exhaust port 8. A reagent impregnatedmesh, such as filter paper, can optionally be situated in condensatedelivery conduit 7 for reagent combination with condensate prior totheir arrival in sample analysis assembly 9. Exhaust port 8 comprises aone-way, low-resistance valve through which air, in which the condensatehas been analyzed, is expelled from the device of the invention.

Sample analysis assembly 9 is connected to the outlet of condensingchamber 5 through condensate delivery conduit 7 and is described indetail with reference to FIG. 2A.

Analyzer 10 which is not a component of the disposable device of theinvention, includes a wavelength-filtered light source and photometerbulb, for measuring the specific absorbance of reacted reagent in sampleanalysis assembly 9, and a computer both for calculating and displayingthe corresponding nitrogen oxide content of the sample in sampleanalysis assembly 9 and for calibrating the absorbance/concentrationrelationship based on standard samples is shown as an association ofconventional elements in essentially block form. Elements 1 through 9 ofthe device of the present invention form a disposable unit which isstored under refrigeration, preferably frozen, until used. In contrast,analyzer 10 is not disposable and is maintained at room temperature.

The disposable unit comprising elements 1-9 is removed from frozenstorage and placed as a unit in connecting housing 11, which is attachedto analyzer 10 in such a way that sample analysis assembly 9 fits snuglyinto connecting housing 11. Connecting housing 11 serves to hold thedisposable and non-disposable units together at the appropriateorientation, and to prevent the subject user's hand from becomingaffected by the cold of the disposable device while holding theapparatus during gas sampling. Sample analysis assembly 9 is surroundedby a thermostatically controlled heating coil 12 attached to connectinghousing 11 and analyzer 10 which brings the sample and test reagents to25° C. Heating coil 12 is part of the non-disposable unit.

Referring now to FIG. 2A, illustrated in hatched line is the outlet ofcondensate delivery conduit 7, which is in enclosed fluid communicationwith sample analysis assembly 9. Sample analysis 9 comprises contiguouswith a round, hard-plastic clear or translucent analysis chamber 13, apliable reagent chamber 14 and pliable element 15. Condensate deliveryconduit 7 and sample analysis assembly 9 are brought into juxtapositionand are connected by pliable element 15, which is a soft-plasticcomponent that extends beyond the diameter of condensate deliveryconduit 7. At the bottom of analysis chamber 13 is an opening 16 inwhich is positioned a fluid-permeable membrane or a thin, plasticrupturable membrane 18.

Reagent(s) for colorimetric nitrogen oxide salt analysis could be adiazo reagent, or such a reagent in conjunction with nitrate reductaseand appropriate cofactors to enable both nitrite and nitrate to bemeasured simultaneously. Other reactions, such as reduction of nitriteand nitrate to nitric oxide in vanadium chloride, followed by reactionwith oxyhemoglobin to achieve a colorimetric shift in the Soret band,and including heterolytic cleavage of S—NO bonds with mercuric chloridefollowed by diazo analysis could also be employed.

When exhaled, the condensate of exhaled air passed into condensatedelivery conduit 7 and then into analysis chamber 13. Thereafter, theuser squeezes pliable element 15, using pincer 17 (shown in FIG. 2B).When pincer 17 is closed against pliable element 15, this causes thevolume of reagent chamber 14 to decrease such that reagent therein isforced upwardly through the opening 16 and permeable or rupturablemembrane into analysis chamber 13 to combine and react with thecondensate. Thereafter, the reaction product of this chemical reactionis analyzed by colorimetry using the non-disposable colorimeter whichpasses a beam of light through a fixed path of analysis chamber 13.

The distal end of the condensing chamber tube(s) 5 is attached to acollecting apparatus comprising condensate delivery conduit 7 whichutilized gravity to trap condensed fluid. At the bottom of this trap isflexion element 15 and the clear plastic analysis chamber 13 in whichthe sample is sequentially warmed, sealed, reacted with colorimetricreagents from reagent chamber 14 and analyzed spectrophotometrically bythe non-disposable colorimeter through the analysis chamber 13.

The patient breathes comfortably in and out through the mouthpiece 1.Lung fluid vapor collects on the inner surface of the inner tube(s) 5 ofthe condensing apparatus starting immediately. Gravity carries thelarger droplets down the tube(s) 5, these droplets recruiting othersmall droplets on their trip to the condensate delivery conduit 7distally.

Alternatively, after a fixed period of tidal breathing, the condensedfluid can be expressed down the inner tube 5 with a device similar to asyringe plunger. Aqueous phase nitrite and nitrate can be measured bystandard colorimetric assays, and can be reasonably quantified by simpletests performed by patients in their homes.

Potential modifications to the system include:

1. Attachment of an assembly distal to the inspiratory portion whichwould contain a fiber paper through which the saliva free exhalate ischannelled. The filter paper would be impregnated with reagents whichreact with nitrogen oxides and subsequently change color. A patientcould breathe through the filter paper for a given period of time andthe degree of color change could be measured against a chart or with asmall colorimetric device.

2. Direct attachment to the distal end of the condensing chamber of afilter paper impregnated with dried reagents which will react with theaqueous phase nitrogen oxides to yield a change in color of the paperwhich can easily be measured in a similar fashion to modification #1above.

3. Direct attachment of a chamber containing liquid reagents throughwhich the exhalate is bubbled, allowing exhaled nitrogen oxides todirectly react with reagents, producing a measurable color change.

4. Attachment of a dehumidifying chamber to the inspiratory portion toeliminate environmental liquid phase nitrogen oxides.

EXAMPLE 1

Expired Nitric Oxide During Tidal Breathing in Humans

Recent insights regarding the use of expired nitric oxide (NO.)concentration measurement as an index of airway inflammation cannot beapplied to children too young to perform vital capacity (VC) maneuvers.We have therefore developed assays which require only tidal breathing.Sequential colorimetric measurements were made during quiet breathing byreacting dried air expirate (DAE) with Griess reagent under acidconditions. Tidal expired gas NO. production rates (V_(NO.)) were alsomeasured by chemiluminescence over 5 minutes using a 765 liter sealedplethysmograph. Eight control subjects and 6 patients with asthma andcystic fibrosis (CF) were studied. DAE NO— was detected in all normalsubject samples with mean (±SD) accumulation curve slope=40±17nmoles/liter/breath (p<0.05 compared with air controls). By comparison,the mean [dissolved nitrogen oxides (NO_(x))] collected in aprecipitation tube (PT) (−80° C.) from size-filtered lung water producedby normal subjects during quiet breathing was 0.8±0.1 μM. TidalV_(NO)/body surface area (specific V_(NO.)[V_(NO.SP)]) was 0.21±0.043μl/min/m² in normal subjects. Consistent with previous reports based onVC measurements, patients with asthma had higher V_(NO.SP) (0.31±0.014μl/min/m², p<0.01) and PT NO_(X) (16±4.0 μM, p<0.005). Further, patientswith CF also had high V_(NO.SP) (0.34±0.84 μl/m²/min, p<0.005). Wereport for the first time 1) the detection of NO— in expired air throughthe use of colorimetric assays; 2) the effort-independent detection ofV_(NO—); and 3) increases in these tidal breath concentrations inpatients with asthma and CF. We speculate that tidal-breathing basedtechniques will have clinical utility both as measures of airwayinflammation applicable to home use and as effort-independent measuresof airway disease in the pediatric pulmonary function laboratory.

EXAMPLE 2

Device Evaluation in Human Clinical Study

Asthma is an inflammatory disease which leads to airway obstruction.Current tests of disease severity are based on measures of obstruction,alterations of which are late findings in asthma exacerbations. Recentstudies reveal that expression of inducible nitric oxide synthase (iNOS)is increased in the airway epithelial and inflammatory cells of patientswith asthma. The activity of iNOS is known to be reflected in highconcentrations of nitric oxide (NO.) gas measured in the expired air ofasthmatic subjects. We studied other aqueous phase NO_(x) in asthmaticaspirate in an effort to develop a simple test for airway inflammationapplicable to home use.

Subjects

6 normal subjects

no smoking history

no history of acute, chronic, or recurrent cardiopulmonary,determatologic, gastrointestinal, or neurologic disease.

13 asthmatic patients

3 or more episodes of reversible bronchospasm

I/E ratio<0.5

FEV₁/FVC<0.75 at study entry

Methods

Saliva free, 0.2 micron particle size-filtered expired vapor wasobtained from subjects breathing quietly through a condensing apparatus.

Nitrite (NO₂—) was assayed colorimetrically using Griess reagent.

Nitrate (NO₃—) was assayed by chemiluminescence after reduction invanadium chloride.

Statistical Methods

Mean NO_(x) concentrations were compared using unpaired t-testing.Results are presented as mean, ± standard error. P values of <0.05 wereconsidered significant.

Results

As shown in FIG. 3, nitrite concentrations were significantly higher inasthmatic subjects (1.877 μM±0.36) than in controls (0.902 μM±0.08)(p<0.01).

As shown in FIG. 4, nitrate concentrations were likewise significantlyelevated in asthmatic subjects (9.48 μM±1.25) relative to controls (5.27μM±0.51) (p<0.01).

Discussion

Tests of airway obstruction, such as the peak flow meter, are currentlythe primary modality for objectively assessing severity of inflammatoryairway disease. These tests have several limitations, including beingeffort-dependent, and thus are not suitable for many children andneuro-muscularly handicapped individuals. They also poorly reflect thedegree of underlying inflammation.

Utilizing recent insights regarding the biochemistry of higher oxides ofnitrogen, we have developed a simple, non-invasive assay for airwayinflammation. Tests such as this could be performed at home, and mayprove to be useful in longitudinal evaluation and management of asthma,including dosing of anti-inflammatory medication.

Conclusion

Aqueous phase nitrogen oxides, measured in condensed exhalate, aresignificantly elevated in asthmatic patients, and can be used todistinguish between normal and inflamed airways.

What is claimed is:
 1. A device for determining a content of higheroxides of nitrogen in exhaled breath condensate comprising: a conduitfor receiving a flow of exhalate, said conduit having an exhalatecondensing portion with an inlet, an outlet and a gas release portcoupled to the outlet, wherein the inlet comprises a mechanicalrespirator; a coolant jacket coaxially surrounding said exhalatecondensing portion; and a sample assay assembly in communication withsaid conduit exhalate condensing portion outlet, wherein the sampleassay assembly comprises: (i) a light transmissive analysis chamberattached to the outlet to receive the exhaled breath condensate, whereinthe light transmissive analysis chamber comprises a reagent entry port,(ii) a reagent chamber in communication with the reagent entry port, and(iii) a pliable element connecting the outlet and the analysis chamberand forming a portion of the reagent chamber, wherein flexion of thepliable element closes communication between the outlet and the analysischamber and contracts the reagent chamber to deliver to the analysischamber controlled amounts of condensate and reagent.
 2. The device ofclaim 1 which further comprises, between the inlet and the exhalatecondensing conduit portion, an inlet assembly comprising a filter forremoving particulate matter from exhaled air which passes therethrough.3. The device of claim 1, which further comprises a coolant material inthe coolant jacket.
 4. The device of claim 1, which further comprises areagent composition for detecting a nitrite or nitrate in the exhaledbreath condensate.
 5. The device of claim 1, wherein the reagent entryport comprises a reagent-permeable membrane.
 6. The device of claim 1,wherein the reagent entry port comprises a rupturable membrane.
 7. Thedevice of claim 1, wherein between the inlet and the outlet is an inletassembly which further comprises a filter for removing ambient nitrogenoxides from exhalate passing therethrough.
 8. The device of claim 1,wherein the exhalate condensing conduit portion comprises a single lumenconduit having a lumen diameter sufficient to cause substantially noresistance to the flow of exhalate theregthrough.
 9. The device of claim1, wherein the exhalate condensing conduit portion comprises a pluralityof conduit tubules having a collective lumen diameter sufficient tocause substantially no resistance to the flow of exhalate therethrough.10. The device of claim 1, which further comprises an inlet assemblybetween the inlet and the outlet for providing one-way ingress ofambient atmosphere to the exhalate condensing conduit inlet.
 11. Thedevice of claim 1, wherein the sample assay assembly comprises a filterpaper or fiber paper.
 12. The device of claim 11, wherein the filterpaper or fiber paper comprises a reagent composition for detecting anitrite or nitrate in the exhaled breath condensate.
 13. The device ofclaim 1, wherein the sample assay assembly comprises at least one liquidreagent.
 14. The device of claim 13, wherein the liquid reagent candetect a nitrite or nitrate in an exhalation condensate sample.
 15. Adevice for determining a content of higher oxides of nitrogen in exhaledbreath condensate comprising: a conduit for receiving a flow ofexhalate, said conduit having an exhalate condensing portion with aninlet, an outlet and a gas release port coupled to the outlet; whereinthe inlet comprises a dehumidifying chamber; a coolant jacket coaxiallysurrounding said exhalate condensing portion; and a sample assayassembly in communication with said conduit exhalate condensing portionoutlet, wherein the sample assay assembly comprises: (i) a lighttransmissive analysis chamber attached to the outlet to receive theexhaled breath condensate, wherein the light transmissive analysischamber comprises a reagent entry port, (ii) a reagent chamber incommunication with the reagent entry port, and (iii) a pliable elementconnecting the outlet and the analysis chamber and forming a portion ofthe reagent chamber, wherein flexion of the pliable element closescommunication between the outlet and the analysis chamber and contractsthe reagent chamber to deliver to the analysis chamber controlledamounts of condensate and reagent.
 16. The device of claim 15, whichfurther comprises, between the inlet and the exhalate condensing conduitportion, an inlet assembly comprising a filter for removing particulatematter from exhaled air which passes therethrough.
 17. The device ofclaim 15, which further comprises a coolant material in the coolantjacket.
 18. The device of claim 15, which further comprises a reagentcomposition for detecting a nitrite or nitrate in the exhaled breathcondensate.
 19. The device of claim 15, wherein the reagent entry portcomprises a reagent-permeable membrane.
 20. The device of claim 15,wherein the reagent entry port comprises a rupturable membrane.
 21. Thedevice of claim 15, wherein between the inlet and the outlet is an inletassembly which further comprises a filter for removing ambient nitrogenoxides from exhalate passing therethrough.
 22. The device of claim 15wherein the exhalate condensing conduit portion comprises a single lumenconduit having a lumen diameter sufficient to cause substantially noresistance to the flow of exhalate theregthrough.
 23. The device ofclaim 15, wherein the exhalate condensing conduit portion comprises aplurality of conduit tubules having a collective lumen diametersufficient to cause substantially no resistance to the flow of exhalatetherethrough.
 24. The device of claim 15, which further comprises aninlet assembly between the inlet and the outlet for providing one-wayingress of ambient atmosphere to the exhalate condensing conduit inlet.25. The device of claim 15, wherein the sample assay assembly comprisesa filter paper or fiber paper.
 26. The device of claim 25, wherein thefilter paper or fiber paper comprises a reagent composition fordetecting a nitrite or nitrate in the exhaled breath condensate.
 27. Thedevice of claim 15, wherein the sample assay assembly comprises at leastone liquid reagent.
 28. The device of claim 27, wherein the liquidreagent can detect a nitrite or nitrate in an exhalation condensatesample.